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Traditional Chinese medicine (TCM), as a unique form of natural medicine, has been used in Chinese traditional therapeutic systems over two thousand years. Active components in Chinese herbal medicine are the material basis for the prevention and treatment of diseases.
Jiao et al Chemistry Central Journal (2018) 12:48 https://doi.org/10.1186/s13065-018-0417-2 Open Access REVIEW Study on the interaction between active components from traditional Chinese medicine and plasma proteins Qishu Jiao, Rufeng Wang, Yanyan Jiang and Bin Liu* Abstract Traditional Chinese medicine (TCM), as a unique form of natural medicine, has been used in Chinese traditional therapeutic systems over two thousand years Active components in Chinese herbal medicine are the material basis for the prevention and treatment of diseases Research on drug-protein binding is one of the important contents in the study of early stage clinical pharmacokinetics of drugs Plasma protein binding study has far-reaching influence on the pharmacokinetics and pharmacodynamics of drugs and helps to understand the basic rule of drug effects It is important to study the binding characteristics of the active components in Chinese herbal medicine with plasma proteins for the medical science and modernization of TCM This review summarizes the common analytical methods which are used to study the active herbal components-protein binding and gives the examples to illustrate their application Rules and influence factors of the binding between different types of active herbal components and plasma proteins are summarized in the end Finally, a suggestion on choosing the suitable technique for different types of active herbal components is provided, and the prospect of the drug-protein binding used in the area of TCM research is also discussed Keywords: Active components, Traditional Chinese medicine, Plasma protein binding, Research methods Introduction Traditional Chinese medicine (TCM) is the summary of practical experience of Chinese people for thousands of years in the fight against disease It is the treasure of Chinese culture and constitutes multi-billion-dollar markets—more than 1500 kinds of herbal medicines are sold as dietary supplements or the raw material of medicines [1] Its active components are the substantial basis for the treatment of various diseases and the related study is also one of the most important parts of the modernization of Chinese herbal medicine Generally speaking, the concentration of the free active (or toxic) components is directly related to the biological effect (or poisoning), and the concentration of the free drugs in plasma is directly related to the concentration in the tissue When drugs are absorbed into the *Correspondence: liubinyn67@163.com School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 102488, China blood, drug-plasma protein binding (PPB) is a common and reversible dynamic process [2] PPB is one of the important parameters of drug efficacy and safety, and the determination of bound fraction is a necessary step in drug discovery and clinical trials [3] It determines the pharmacokinetic and pharmacodynamic characteristics of drugs and influences drug absorption, distribution, metabolism, excretion and toxicity (ADMET) [4, 5] It is generally considered that only free drug can transfer through biological membranes, combine with the appropriate site of action and drive the therapeutic outcome [6] And then it displays the pharmacological and/ or toxicological effects [7] Small molecular substances can be protected from some elimination pathways, such as enzymatic reactions in the liver or blood and glomerular filtration of the kidneys, by forming non-covalent complexes with plasma proteins [8] As a drug reservoir, the bound drug fraction can maintain an effective concentration and prolong the duration of the drug action For the drugs with high affinity for plasma proteins, they © The Author(s) 2018 This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated Jiao et al Chemistry Central Journal (2018) 12:48 generally need a higher dose to reach therapeutic level, have a long half-life and probably increase toxicity Conversely, the drugs with low plasma protein binding affinities are limited in their ability to perfuse tissues and reach the site of action [9] Although many Chinese herbal medicines have been proved to be effective by modern clinical trials and pharmacological studies, their active components and the remedial mechanism are still unclear [10] The pharmacological activities of Chinese herbal medicines are considered to be the combination of multi-components effects, including the interactions of active components with proteins It is well known that a kind of herbal medicine usually contains hundreds of different components [11] There is no doubt that this is a complex and heavy work to elucidate the mechanism of action of these components Therefore, it is extremely valuable to investigate the binding of one or a few active components from Chinese herbal medicine with plasma proteins Plasma proteins involved in drug binding Major drug-binding proteins in plasma are human serum albumin (HSA), α1-acid glycoprotein (AAG) and lipoproteins [12] They have many important physiological functions, for example, mediating osmotic pressure and nutrient delivery, participating in the clot formation and immune response [13] It is generally accepted that acidic drugs display greater affinity for HSA, while AAG is primarily responsible for the binding of neutral and acidic drugs [14] HSA, as the most abundant protein in plasma proteins, is in a position to bind endogenous ligands (e.g., fatty acids, amino acids, hormones, bile acids, metals and toxic metabolites) as well as drugs [15–17] AAG is the second most abundant one, and its endogenous ligands include heparin, serotonin, histamine, steroid hormones and so on [18] Research reporting on drugs binding to lipoprotein is still sparse As the most abundant plasma protein with amazing properties and functions, HSA is the most widely explored protein which is always used as the ligand-biological macromolecules interaction model [19] Through the first crystallographic analyses of HSA, it is revealed that the protein, as a kind of nonglycosylated molecules, consists of 585 amino acids and 35 cysteine residues, forming 17 disulfides and one free sulfhydryl group at Cys34 The classical researches revealed that the atomic structure of HSA consists of three homologous α-helical domains (I–III) each including two subdomains (A and B) [20] The protein has two high affinity drug binding sites, named as Sudlow’s sites in subdomain IIA and IIIA [8] Drug site (subdomain IIA) is composed of three extended sub-chambers and a central zone The inside of the pocket is mainly non-polar molecules Two clusters Page of 20 of polar residues located in the bottom (Tyr 150, His 242, Arg 257) and the entrance (Lys 195, Lys 199, Arg 218, Arg 222) are also identified Drug site is occupied by phenylbutazone and warfarin Drug site (subdomain IIIA) is smaller than site 1, but it can accommodate large molecules, such as ibuprofen and thyroxine In addition, there is another binding site named site 3, to which the digitoxin binds Because of the structural homology with HSA, bovine serum albumin (BSA) is also a common interaction model used for investigating PPB [21] Methods to investigate the interaction between active herbal components and plasma proteins In recent years, with the development of Chinese herbal medicine, researchers have been paying more and more attention to the pharmacological activity of components in herbal medicine, and numerous experimental techniques have been used in the characterization of PPB The work has become increasingly diverse and detailed by the application of spectroscopy, chromatography, thermodynamics, electrochemistry and other techniques The principle and the detection methods of current analysis tool have been introduced in several theses [8, 19, 22] In this article, a brief introduction of common methods is given, and the applications of techniques used in the investigation of the interaction between active herbal components with plasma proteins are described in detail Membrane technology Equilibrium dialysis (ED) ED, combined with highly-sensitive assay, such as high performance liquid chromatography (HPLC) and mass spectrometry (MS), is regarded as the gold standard to determine protein binding rate The working principle of ED is that the small drug molecules could be separated from protein solution by semipermeable membrane The small drug molecules could pass through the semipermeable membrane until the dialysis reaches equilibrium, while the drug-protein complexes are retained in the dialysis bag The binding rates of drug molecules with plasma proteins can be calculated by measuring the concentrations of small molecules in the solution on both sides ED is an easy, economical, practical method and can eliminate the possible effect of non-specific binding [23, 24] In recent years, ED has been widely used in the multi-component drug research in Chinese herbal medicine Liu et al investigated the effects of sinomenine on the therapeutic action of paeoniflorin in the treats-rats by an equilibrium dialysis assay in vitro [25, 26] The results showed that the protein binding ability is not influenced when they are administrated simultaneously Wang et al used a kind of dialysis sampling on-line coupled with Jiao et al Chemistry Central Journal (2018) 12:48 HPLC (DS-HPLC) to monitor the interactions of multicomponents in danshen (Salvia miltiorrhiza) injection with BSA [27] The five components (danshensu, protocatechuic acid, protocatechuic aldehyde, caffeic acid and ferulic acid) in danshen injection had suitable binding degrees with BSA Talbi et al found that wogonin had a very high protein binding degree (over 90%) with rat plasma [28] But ED also has disadvantages in some ways, including a long time for balancing, the strict control of the pH of plasma and buffer solution, dilution effect and Gibbs–Donnan effects, etc [19, 29–31] In recent years, the equilibrium dialysis devices based on the 48-well and 96-well plates have been used in the plasma protein binding studies [32] The unique design of the device increases the surface area-to-volume ratio and offers the possibility of reducing equilibration times and higher assay throughput Compared with traditional equilibrium dialysis, this device also has many advantages including easy-to-clean, reusability, the reduction of the drug nonspecific absorption and capability of being automated As the early screening tools for drug research, rapid equilibrium dialysis (RED) device and parallel artificial membrane permeability assay (PAMPA) are two major in vitro models based on Teflon base plate The RED device comprises replaceable tube inserts and a 48-well Teflon base plate Each insert is divided into a buffer compartment (white) and a plasma compartment (red) by a semipermeable membrane at molecular weight cut-off (MWCO) = 8000 Each plate could be sealed with sealing tape and self-adhesive lid The volume of the insert should be checked to guarantee the little to no volume change occurred [33–35] Kim et al developed a RED device combined with LC–MS/MS method to quantify the acacetin in human plasma [36] The results showed a concentration-independent and extensive protein binding of acacetin in human plasma The general PAMPA plate system consists of an acceptor compartment (96-well filter plate) and a donor compartment (96-well receiver plate) [37] Each well of the 96-well microfiltration membrane is filled with 10 μL of the artificial membrane solution which is made of film-forming material dissolved in organic solvent The 96-well filter plate will be placed on the receiver plate to allow the artificial membrane to touch the donor fluid And thus the system forms a sandwich structure: the bottom is the donor liquid of the sample, and the drug molecules diffuse from the donor tube into the upper receptor tube through the artificial membrane When the diffusion is completed, the receptor fluid and the donor fluid can be used to make quantitative analysis [38, 39] Singh et al investigated the blood uptake characteristics, protein binding, pharmacokinetics and metabolism Page of 20 of formononetin by this system Formononetin had high protein binding rate, and the rapid absorption of which might due to the high permeability and lipophilicity [40] Ultrafiltration Ultrafiltration is a popular alternative of ED and a better choice for the clinical pharmacokinetic and pharmacodynamic studies of new drugs [41] Similar to ED, it utilizes semipermeable membrane to separate the device into two chambers Driven by the pressure difference or centrifugation (approximately 2000×g), the drug molecules diffuse through the semipermeable membrane Because this method achieves the rapid separation of small molecules in plasma, the work efficiency is greatly increased [42] Ultrafiltration is more suitable for highly lipophilic compounds, and it, in combination with HPLC, GC–MS, LC-IT-TOF–MS, RRLC-ESI–MS–MS and other high sensitivity detection methods, has been applied to determine the plasma protein binding rate of active herbal components [43–50] In ultrafiltration, the concentration polarization, which is caused by the diffuse direction of the small molecules, is perpendicular to the ultrafiltration membrane It will compromise the protein-binding equilibrium and affect the determination of free drug concentration Li et al developed a novel and practical method based on hollow fiber centrifugal ultrafiltration (HFCF-UF) combined with HPLC to determine the plasma protein binding of three coumarins in human plasma [51] The device was made of a glass tube, in which a U-shaped hollow fiber was placed Therefore, the direction of molecular diffusion was completely parallel to the membrane The binding rates of bergenin, daphnetin, and scopoletin determined by this method were 52.7–53.5, 56.7–58.0 and 59.0–60.1% respectively, which were consistent with the results of the equilibrium dialysis method Compared with the classical method, HFCF-UF has higher precision and accuracy and simpler sample preparation procedure Microdialysis Microdialysis was originally used to determine the free adenosine levels in the brain of rats [52] In recent years, it has become an important technique for direct determination of the free drug concentration in the body’s plasma, tissue and other physiological fluids The key of this technique is the probe with a semipermeable membrane which has a molecular mass cut-off ranging from 5000 to 50,000 Da [53] The biggest advantage of microdialysis is the real-time sampling and on-line analysis in a condition that hardly interfered with the normal life activity of animals [54] With this method, we can continuously measure the concentration of unbound drug over time in vivo [55] Another advantage of microdialysis is Jiao et al Chemistry Central Journal (2018) 12:48 the convenience for automation that hyphenated with many sensitive analytical techniques like HPLC, capillary electrophoresis (CE), nuclear magnetic resonance (NMR), etc [56] Microdialysis has many features in the field of traditional Chinese medicine The most prominent feature is the ability to simultaneously investigate the interaction of multi-components in Chinese herbal medicine or compound prescription with plasma proteins, and thus finding the potential active components [57] Qian et al found that chlorogenic acid, luteolin-3-O-glucoside and 4,5-di-O-caffeoyl quinic acid might compete for the same binding sites and caffeic acid and rutin had synergistic effects in Flos Lonicerae Japonicae [58] Wen et al found that four compounds (chlorogenic acid, calycosin-7-O-βd-glucoside, ferulic acid and calycosin) in Danggui Buxue Decoction had suitable binding degrees with human plasma proteins [10] These compounds had been proven to be the active components in the prescription Guo et al found that compound I and compound M identified in Rhizoma Chuanxiong had the similar binding degrees to HSA as two known active compounds, ferulic acid and 3-butylphthalide [59] They thought compound I and compound M might be the potential active compounds The online coupling of microdialysis with sensitive and selective analytical systems has great value and potential in screening the effective components from Chinese herbal medicine Centrifugation Other than the membrane techniques like ED and UF, ultracentrifugation (UC) techniques separate the free drug molecule from the drug-protein complex by high gravitational force (625,500 g) Small molecules and proteins have different density or sedimentation rate in centrifugal force field After centrifugation, the drug molecules combined with high density plasma macromolecules will rapidly subside to the bottom, while the free fraction can be quantitated in the supernatant of the centrifuge tube [8, 60] UC has several advantages such as the lack of Gibbs–Donnan effects and nonspecific adsorption, adoptability for high molecular weight and lipophilic compounds [61] But the limit factors, like the expensive equipment and the low throughput caused by the relatively smaller number of samples that can be processed at one time, restrict the application of UC techniques Li et al removed the plasma proteins by ultracentrifugation and measured the concentration of syringopicroside in serum by HPLC after injection of low, medium and high doses [62] The results showed that syringopicroside was a medium plasma protein binding drug and the binding rate was not dependent on the doses Page of 20 Extraction methods Solid phase microextraction (SPME) SPME is a simple and effortless technique to determine free drug concentration [63] It was developed as a convenient method for volatile organic compounds in the early 1990s Because of its simplicity, SPME has been used to monitor the metabolites, ligand–protein binding, toxicity and permeability of drugs, and metabonomics of volatile or semivolatile compounds Basic theory of this technique is that the solid support, which is hydrophobic and dispersed with extracting phases, is exposed to the test sample for a definite period of time [64] Then, the enriched drug molecules in the extraction phase are rapidly and completely separated into the analytical instruments by high temperature or solvent elution methods SPME fiber is an optical glass fiber which is evenly coated with a polymer coating [65] Because of the non-depleting extraction mode, SPME is a particular suitable technique for drug-protein binding studies [66] The development of biocompatible coating makes SPME can investigate complex biological samples for any binding equilibriums [64, 67] The relative high accuracy and sensitivity, no need to use organic solvents and possibility to automate are the main advantages of SPME But the fouling formed of protein-fiber binding may lead to erroneous estimate of the concentration in the fiber coating [63, 65] SPME has been used in investigating the interaction between active components in TCMs and plasma proteins [68–70] Volatile oil widely exists in traditional Chinese medicine derived from plants It is well known that there are 136 genera of 56 families in China containing volatile oil In addition to volatile oil, there are many aromatic substances of Chinese medicine, such as musk, bezoar and borneol These components are complex, volatile and insoluble in water Therefore, conventional methods are difficult to determine the binding degrees of these components with plasma proteins Headspace-SPME, in which the extraction fiber is placed in the upper space of the samples, is more suitable for the determination of these components The extraction head of headspace-SPME does not touch the sample, and thus avoids the matrix effect Hu et al developed a headspace negligible-depletion extraction mode (nd-SPME) coupled to GC method to investigate the noncovalent interaction of borneol with HSA [71] The method was simple, sensitive, rapid and could overcome the drawback of losing volatile components in the binding or transfer process Hollow fiber liquid–liquid phase microextraction (HF‑LLPME) HF-LLPME is an inexpensive sample preparation method to investigate the drug-protein binding under physiological conditions without disturbing the equilibrium Jiao et al Chemistry Central Journal (2018) 12:48 between drugs and proteins [72] In microextration system, the polypropylene hollow-fiber membrane is filled with 15–25 μL of extraction solvent and placed into the mixture of drug and protein When small molecule drugs establish distribution equilibrium between bulk aqueous phase and organic phase, the unbound concentration of drugs can be determined by analytical instrument [73, 74] This method allows simultaneous determination of multi-components Compared with the traditional liquid–liquid extraction (LLE), HF-LLPME allows the sample under vigorous stirring conditions and requires less organic solvents Therefore, the method reduces the analysis time of drugs transferred across the membrane HFLLPME has potential to determine drug-protein binding of active components from TCMs in the complex sample matrices Hu et al investigated the interaction of four furocoumarin and two alkaloid compounds with BSA by HF-LLPME combined with HPLC [75, 76] The results demonstrated that HF-LLPME is a simple, rapid and effective method for characterizing drug-protein binding parameters without separation Chromatographic methods High performance affinity chromatography (HPAC) HPAC is a kind of adsorption chromatography which uses a biologically related agent as stationary phase [77] As one of the most effective methods that separate and purify the biological macromolecules, HPAC is based on the specific reversible interaction between the target protein and the immobilized ligand HPAC immobilizes the proteins onto a support and injects the interacting solute into the column The drugs with high affinities will be eluted later than low-affinity drugs because of the strong interaction [78] The method has been coupled with HPLC to determine the binding of drugs and various proteins such as HSA, AGP and lipoproteins in plasma [79] Many reports have demonstrated that the allosteric interactions and displacement effects seen on HSA columns are similar to those observed for soluble HSA [80, 81] Compared to the traditional methods, HPAC has many advantages such as automation, high precision, speed, specificity and the ability to work with small amounts of a target solute [82, 83] But some problems still need to be solved, such as the short service life of the column and the high standards of the preparation of fillers For complex research objects, such as Chinese herbal medicines, HPAC could eliminate the interference of a large number of inactive impurities due to the specificity and selectivity of the stationary phase in combination with the active component Cai et al detected the binding rates of puerarin and goitrin with HSA by a HSA column [84] The results were consistent with those obtained by ultrafiltration method and demonstrated Page of 20 that HPAC method was a reliable technique HPAC is often applied to investigate the competition displacement in different active herbal components with plasma proteins Lei et al investigated the competition interaction of ferulic acid and paeonol with HSA by HPAC [85] The results demonstrated that ferulic acid and paeonol competed for binding to the indole site (site 2) and the main force was deduced to be hydrogen bonding according to the thermodynamic parameters Capillary electrophoresis (CE) CE is a series of related techniques that the separation processes are happened in narrow bore capillaries under the force of electric field [86] It is a powerful analytical tool that is widely used in the analysis of small organic molecules, inorganic ions and biopolymers [87] In the years past, CE has become a hit for drug-protein interaction measurements because of low sample requirements and consumption, simplicity, short analysis times, high sample throughput and high separation efficiencies [5, 88] There are several modes of electrophoresis to investigate the drug-protein binding, including affinity CE (ACE), vacancy peak (VP), Hummel–Dreyer method (HD), frontal analysis (FA) and zone migration CE (CZE) [89] Among them, ACE, FA and CZE have the same advantages: (1) only a small number of proteins and drugs are required; (2) all interacting components can be investigated in free buffer solution at physiological conditions; (3) binding constants of multi-components can be simultaneously estimated Therefore, these methods are suitable for the study of some Chinese herbal medicines which are chemically complex and expensive [90–93] In recent years, with the development of microdialysis in the field of medicine, CE combined with microdialysis techniques has been used in pharmacokinetics research [94, 95] It combines the characteristics of continuous, dynamic sampling in microdialysis and less sample volume in CE The method could objectively analyze the drug-protein binding behavior of specific drugs under physiological and/or pathological conditions Although there are few reports about the research on CE combined with microdialysis techniques in the field of TCMs, there is no doubt that it is the best choice if you want to study the change of multi-components in Chinese herbal medicine and plasma protein binding in disease states Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‑PAGE) SDS-PAGE, which was proposed by Laemmli in 1970, is a charming and powerful tool for protein characterization [96] The principle of SDS-PAGE is the positive correlation between electrophoretic mobility of protein and the molecular mass [97–99] SDS, a kind of anionic Jiao et al Chemistry Central Journal (2018) 12:48 detergent, could denature original proteins, eliminate protein’s original surface charge and destroy the structure And then the SDS-protein complexes are formed The advantages of SDS-PAGE are simplicity, less analysis time and excellent repeatability However, because of the large errors and low resolution of SDS-PAGE, the method cannot reflect the binding degree of drug and the application is rare Kaldas et al identified the irreversible binding between oxidized quercetin and protein by a radioactively labelled drug and SDS-PAGE The result showed that quercetin oxidized by hydrogen/peroxidase covalently links to proteins and with particularly high affinity for HSA [100] Spectroscopic methods The main spectroscopic methods of the interaction between active herbal components and plasma protein Spectroscopic methods are based on the change of spectroscopic properties of proteins in ligand–protein binding processes The information of drug-protein binding can be obtained without separation Fluorescence spectroscopy Fluorescence spectroscopy is the most widely used and powerful spectroscopic technique for gaining the information about the binding of drug and plasma proteins because of its accuracy, sensitivity, rapidity and usability [101, 102] Because of the existence of aromatic, such as tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe), serum proteins are considered as endogenous fluorescent substance When 295 nm is selected as the excitation light source, endogenous fluorescence is all from the Trp residue [103] When small molecule drugs interact with proteins, they are often able to decrease the fluorescence intensity or quench the intrinsic fluorescence of proteins Synchronous fluorescence spectrum, which can be obtained by simultaneous scanning excitation wave and emission wave, could determine the emission spectra of Tyr and Trp Threedimensional fluorescence spectroscopy, a kind of new fluorescence analytical technologies developed in the past 20 years, can visually show the microenvironment and conformational changes under different conditions of Trp in protein molecules UV–Vis absorption spectroscopy UV–Vis absorption spectroscopy is another widely used technique to investigate drug-protein binding Inherent ultraviolet absorption of plasma protein is mainly due to the absorption of light generated by the n–π* transition in indolyl group of Trp, the phenol group of Tyr and the phenyl group of Phe The changes of the peak intensity and position of two characteristic absorptions could reflect the conformational change of proteins Page of 20 Fourier transform infrared spectroscopy Fourier transform infrared spectroscopy (FT-IR) is one of the popular techniques for the structural characterization of proteins The most important advantage of FT-IR compared with other methods is the extensive applicability of any biological system in a wide variety of environments [104, 105] The characteristic absorption peaks of amide groups of proteins are the most valuable ones for the study of protein secondary structure Circular dichroism spectroscopy Circular dichroism (CD) spectroscopy is based on the different absorption of the left and the right circularly polarized light by optically active groups of proteins The CD spectra of serum proteins are generally divided into two wavelength ranges—178–250 nm for the far-ultraviolet CD spectrum and 250–320 nm for the near-ultraviolet CD spectrum Extrinsic Cotton effect is used to represent the change of the normal CD spectrum in the binding of ligands to HSA The far-ultraviolet CD spectrum, the most commonly used spectrum in protein study, could reflect the protein secondary structure information The peak in the near-ultraviolet region is sensitive to reflect the subtle changes in the conformation of the serum protein Surface plasmon resonance (SPR) Surface plasmon resonance (SPR), which can monitor the formation and dissociation of the drug-protein complex in real-time and obtain the equilibrium (KD) and kinetic (kon and koff) data for the interaction, is one of the most excellent optical biosensor technologies [106–108] The conventional SPR device requires a biomolecule to be immobilized on a sensor chip The sensor chip can monitor the change of refractive index that occurs at the surface of the complexes during form or break process in the binding reaction [108–110] Another partner in solution is placed together with the sensor Fabini et al developed a sensor chip whose serum albumins were covalently bound to the carboxymethyl dextran layer of the sensor chips through its primary amine groups by an amine coupling reaction [111] The result indicated that cucurbitacins were able to modulate the binding of biliverdin and serum albumins Compared with the traditional analytical methods or means, SPR has much salient features such as free label detection, real-time dynamic analysis, non-destructive testing, high sensitivity and larger detection range [112, 113] Shi et al developed a rapid, continuous and effective method to identify the multi components from Radix Astragali which were bound to HSA by a SPRHPLC–MS/MS system [114] The data of reverse ultrafiltration assay showed a good agreement with SPR SPR has become a popular technique to study DNA–DNA, antibody-antigen, protein–protein interaction and the Jiao et al Chemistry Central Journal (2018) 12:48 interaction between drugs and specific cellular receptor proteins, key genes, proteases and other disease-related biomolecules Besides, there are several commonly used spectra like mass spectrometry (MS), nuclear magnetic resonance (NMR) spectrum, resonance light scattering (RLS) and surface-enhanced Raman scattering spectroscopy Several spectra are generally used together to study the drugprotein binding and could give more comprehensive data and results The main research contents of spectroscopic methods of the interaction between active herbal components and plasma protein What we can learn from the result of the spectroscopic methods about the binding between plasma proteins and active herbal components include judging the mechanisms of fluorescence quenching, calculating the binding constant, the number of binding sites, the distance between Trp and drug molecule and thermodynamic parameter, determining the binding site, binding forces and change of protein’s secondary structure, etc Mechanisms of fluorescence quenching The effect of active herbal components on the intrinsic fluorescence of serum albumin can be divided into fluorescence quenching and fluorescence sensitizing In most cases, fluorescence quenching is the main one The mechanism of fluorescence quenching can be classified as dynamic quenching and static quenching The reason for the static quenching is the formation of non-fluorescent complex between the fluorescent molecules in the ground state and quencher [101] So that the fluorescence spectra of the static quenched fluorescent molecules change The dynamic quenching is caused by the collision of the fluorescent molecules in the excited state with the quencher After collision, the fluorescent molecules return to the ground state, so that the fluorescence spectra of the dynamic quenched fluorescent molecules not change The mechanism of fluorescence quenching can be determined by the following points [115] Firstly, in the Stern–Volmer equation, the value of Kq is about 109–1010 L (mol s)−1 If Kq calculated from Ksv and τ0 is much larger than this range, it means that the binding is not diffusion control and the mechanism of fluorescence quenching is static quenching Conversely, the mechanism may be dynamic quenching The Kq of delphinidin-3-O-glucoside at 298 K was 6.163 × 1012 L mol−1s−1, which was much higher than the maximum diffusion collision quenching constant value (2.0 × 1010 L mol−1s−1) It illustrated that the interaction of delphinidin-3-O-glucoside with BSA occurred by the static quenching [116] Page of 20 Secondly, when the dynamic quenching occurs, the UV–Vis absorption spectra of fluorescent molecules not change In the event of static quenching, the changes occur on the UV–Vis absorption spectra of fluorescent molecules HSA had an absorption peak approximately at 280 nm on the UV–Vis absorption spectra The increasing neohesperidin dihydrochalcone concentration decreased the absorption peak of HSA and a slight blue shift could be observed These evidences showed that the interaction between neohesperidin dihydrochalcone and HSA belonged to static quenching [117] Thirdly, dynamic quenching relies on molecular diffusion The temperature rise increases the diffusivity of the molecules and the possibility of molecular collision So the quenching constant increased with temperature On the contrary, the increase of temperature may reduce the stability of non-fluorescent complex, thereby reducing the degree of static quenching The value of Ksv of ferulic acid was 3.818 × 104, 3.912 × 104, and 4.881 × 104 at 25, 35 and 45 °C The trend that the quenching constant increased with the increase of temperature indicated that the interaction of ferulic acid with HSA was influenced by diffusion [118] And, fourthly, in the case of static quenching, quenching does not change the lifetime of the excited state of fluorescent molecules: τ0/τ = Whereas in the case of dynamic quenching, the presence of the quencher reduces the lifetime of fluorescence: τ0/τ = F0/F Yang et al found that the increasing concentration of paclitaxel hardly changed the lifetime of HSA (from 5.58 to 5.47 ns) and the quenching followed a static mechanism [119] But for some active herbal components, the static and dynamic procedure may exist simultaneously Cheng et al investigated the interaction of tetrandrine with BSA and HSA The trend that the values of Ksv increased with the increasing temperature indicated that the interaction belonged to dynamic quenching [120] But the UV– Vis spectra data and the higher Kq ~ 1013 L mol−1s−1 at 298 K showed the formation of complex Therefore, a combination of the static and dynamic quenching played an important role in the interaction of tetrandrine with BSA and HSA Similarly, Gao et al found an increase of absorbance band intensity on the UV–Vis spectra when the concentration of syringin was increased in HSA [121] However, the value of K increased with the increasing temperature Therefore, they thought that the quenching mechanism of HSA by syringin was dynamic quenching, while static quenching could not be ignored Binding constant and the number of binding sites Binding constant and the number of binding sites can be calculated by Stern–Volmer equation, modified Stern–Volmer equation, Lineweaver–Burk equation, Benesi–Hidebrand Jiao et al Chemistry Central Journal (2018) 12:48 equation, Benesi–Hidebrand equation and multiple binding sites equation Stern–Volmer equation is the most well-known formula which is used to calculate binding constant and the number of binding sites and could apply to study the fluorescence quenching mechanism Both static quenching and dynamic quenching process follow this equation [19] Modified Stern–Volmer equation could reduce the effect of other light in the fluorescence experiment on the measured value [122, 123] When the linearity of the Stern–Volmer equation is not ideal, the Lineweaver–Burk equation can be used But Matei et al predicted slightly higher K values by this model than classical Scatchard equation in the investigation of the kaempferol-HSA complex [124] They thought that in fact here K represented quenching constant which was used to describe the binding efficiency of the quencher to the fluorescent molecules, but not the binding constant This equation applies to the system with only one binding site If the small molecule ligand has fluorescence, its fluorescence intensity increases as it interacts with the protein Bhattacharya et al modified the Benesi–Hidebrand equation to escape this interference [125] This equation is suitable for the active herbal components which have auto-fluorescence [126] For the multiple binding sites system, Zhang et al proposed a multiple binding sites equation that could calculate the binding constant and the number of binding sites at the same time [127] The binding constants and the number of binding sites of N-transp-coumaroyltyramine, 3-trans-feruloyl maslinic acid, four flavonoid aglycones (baicalein, quercetin, daidzein, and genistein) and their monoglycosides (baicalein, quercitrin, daidzin, and puerarin, genistin) were all calculated by this equation [128–130] It is noteworthy that all the active herbal components using this equation must follow static quenching Thermodynamic parameter and binding forces The binding forces between small molecules and proteins include hydrophobic interactions, electrostatic interactions, hydrogen bonds and van der Waals forces [131] According to the thermodynamic parameters, the type of binding forces can be roughly determined The change in enthalpy (∆H) can be considered as a constant when the temperature changes a little Then the values of enthalpy changes and entropy changes (∆S) can be calculated from van’t Hoff equation Ross et al thought that the type of binding forces can be determined by the sign and magnitude of the thermodynamic parameter [132] The relationship between thermodynamic parameters and binding forces are shown in Table 1 However, the structure of HSA is very complex and usually there are multiple forces between small molecules and proteins in the actual reaction system For example, Page of 20 Table 1 The relationship between thermodynamic parameters and binding forces Thermodynamic parameter Binding force ∆S > 0 May be hydrophobic and electrostatic interactions ∆S 0, ∆S > 0 Hydrophobic interactions ∆H